System and method for detecting and monitoring pathogens

Abstract

Disclosed herein are systems, methods and kits for detecting and monitoring pathogens, such as bacterial, fungal and viral foodborne pathogens.

1 . A method for detecting, quantitating and/or monitoring pathogens, comprising:
 capturing one or more live pathogens from a contaminating matrix within a sample by aptamer-based capture or antibody-based capture; and   releasing the one or more captured live pathogens from aptamers or antibodies, thereby allowing the one or more live pathogens to be detected, quantitated and/or monitored without requiring cell culture.   
     
     
         2 . The method of  claim 1 , wherein the one or more live pathogens is one or more disease producing organism, such as bacteria, fungi, protozoa and/or worms. 
     
     
         3 . The method of  claim 1 , wherein the one or more live pathogens is one or more pathogens in a sample from food, water, environment, soil, plant, animal, insect, or human. 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein capturing one or more live pathogens comprises applying the sample to a pathogen-isolation column containing multiple beads of one or more sizes. 
     
     
         5 . The method of  claim 4 , wherein cross-sectional area of the pathogen capture column is constant. 
     
     
         6 . The method of  claim 4 , wherein cross-sectional area of the pathogen capture column varies. 
     
     
         7 . The method of  claim 4 , wherein cross-section of the pathogen capture column is of a uniform shape, such as of a circle, oval or polygon. 
     
     
         8 . The method of  claim 4 , wherein cross-section of the pathogen capture is a non-uniform shape. 
     
     
         9 . The method of any one of  claims 1 - 8 , wherein magnesium is present at pathogen capture at a concentration sufficient to increase aptamer melting temperatures to result in stable secondary structure formation while in the presence of less than 100 mM sodium and less than 20 degrees Celsius. 
     
     
         10 . The method of  claim 9 , wherein the magnesium concentration is more than 0.1 mM. 
     
     
         11 . The method of any one of  claims 4 - 10 , wherein bead surface of each of the multiple beads comprises of a material that has been modified for aptamer or antibody attachment. 
     
     
         12 . The method of any one of  claims 1 - 11 , wherein capturing one or more live pathogens further comprises applying the sample to a pre-filter container containing beads of same size or smaller than pathogen-isolation column prior to the pathogen-isolation column containing multiple beads of one or more sizes. 
     
     
         13 . The method of  claim 12 , wherein the beads of the pre-filter container have non-fouling surface properties. 
     
     
         14 . The method of  claim 12  or  13 , wherein capturing one or more live captured pathogens from aptamers or antibodies, comprises:
 optional conditioning of the pre-filter container and capture column by flowing liquid through the pre-filter container and capture column. 
 
     
     
         15 . The method of any one of  claims 1 - 14 , wherein the sample is a pathogen sample. 
     
     
         16 . The method of any one of  claims 1 - 15 , further comprising washing the one or more live captured pathogens prior to releasing the live captured one or more pathogens to remove substances not specifically bound. 
     
     
         17 . The method of  claim 16 , wherein substances not specifically bound comprise non-targeted organisms and nucleic acid amplification inhibitors. 
     
     
         18 . The method of any one of  claims 1 - 17 , wherein releasing the one or more live captured pathogens from aptamers or antibodies, comprises washing the captured one or more live pathogens with a release buffer. 
     
     
         19 . The method of  claim 18 , wherein releasing the one or more captured live pathogens from aptamers or antibodies is performed by using a flow rate equal to or higher than the flow rate used to capture the one or more pathogens. 
     
     
         20 . The method of  claim 19 , wherein the flow rate for releasing the one or more captured live pathogens from aptamers or antibodies is at least 2× higher than the flow rate for capturing the one or more pathogens. 
     
     
         21 . The method of any one of  claims 1 - 20 , wherein releasing the one or more live captured pathogens from aptamers or antibodies, comprises an air gap prior to washing the captured one or more live pathogens with a release buffer. 
     
     
         22 . The method of  claim 21 , wherein the released live pathogens are optionally collected in liquid in a vented bubble trap acting as a reservoir and acting to remove air prior to the one or more live pathogens in liquid are flowed through a filter to collect the released one or more pathogens. 
     
     
         23 . The method of  claim 22 , wherein the vented bubble trap comprises at least one inlet port, at least one outlet port, and a vent to air. 
     
     
         24 . The method of  claim 23 , wherein the vent optionally comprises one or more sensors that permit feedback control of volume of liquid pumped into the vented bubble trap. 
     
     
         25 . The method of  claim 24 , wherein the one or more sensors comprise one or more liquid level sensors capable of detecting liquid level and/or one or more bubble sensors capable of detecting bubbles in the liquid. 
     
     
         26 . The method of any one of  claims 1 - 25 , wherein the method is aptamer-based and aptamers are used to specifically sequester pathogens away from the contaminating matrix. 
     
     
         27 . The method of any one of  claim 26 , wherein the aptamer contains DNA, RNA, PNA, peptide or other natural or synthetic molecules 
     
     
         28 . The method of any one of  claims 1 - 27 , further comprising detecting, quantitating and/or monitoring pathogens by performing nucleic acid detection following releasing captured live pathogens. 
     
     
         29 . The method of  claim 28 , wherein nucleic acid detection comprises detecting DNA or RNA. 
     
     
         30 . The method of  claim 28  or  29 , wherein nucleic acid detection comprises performing polymerase chain reaction, isothermal amplification, hybridization detection, and/or sequencing. 
     
     
         31 . The method of any one of  claims 1 - 30 , wherein one or more steps is performed by automation. 
     
     
         32 . The method of any one of  claims 1 - 31 , further comprising sanitizing after performing the method of detecting, detecting, quantitating and/or monitoring pathogens. 
     
     
         33 . The method of  claim 32 , wherein sanitizing comprises employing a sanitation system comprising a cartridge and a sanitation solution for sanitation prior to or following use of system for detecting, monitoring or quantitating one or more pathogens. 
     
     
         34 . The method of any one of  claims 1 - 33 , further comprising applying a sample temperature by one or more cooling or heating elements to transfer the temperature to the sample prior to pathogen capture. 
     
     
         35 . A system for detecting, quantitating and/or monitoring pathogens, comprising:
 a sample input source for holding the sample prior to processing;   a sample straw coupled to the sample input source;   a pump coupled to the sample straw for providing the sample for processing; and   one or more sensors to detect when sufficient sample volume is collected or sample volume is no longer detectable.   
     
     
         36 . The system of  claim 35 , wherein the one or more sensors comprises one or more liquid level sensors capable of detecting liquid level and/or one or more bubble sensors capable of detecting bubbles in the liquid. 
     
     
         37 . The system of  claim 35  or  claim 36 , further comprising a process for notifying an insufficient sample volume being acquired. 
     
     
         38 . The system of any one of  claims 35 - 37 , further comprising a pathogen capture system coupled to the sample straw, wherein one or more pathogens are captured and a method to analyze the captured pathogen. 
     
     
         39 . The system of any one of  claims 35 - 38 , wherein the sample input source is a temporary or permanent bag or reservoir. 
     
     
         40 . The system of any one of  claims 35 - 39 , wherein the straw is temporary or permanent. 
     
     
         41 . The system for of any one of  claims 35 - 40 , further comprising a sanitation system for sanitation prior to or following use of the system. 
     
     
         42 . The system of  claim 41 , wherein the sanitation system comprises a sanitation cartridge and sanitation solution for sanitation prior to or following use of system for detecting, monitoring or quantitating one or more pathogens. 
     
     
         43 . A method for detecting, quantitating and/or monitoring pathogens, comprising:
 capturing one or more foodborne pathogens from a contaminating matrix within a sample by aptamer-based pathogen capture or antibody/antigen pathogen capture; and   releasing the one or more captured foodborne pathogens from aptamers or antibodies, thereby allowing the one or more foodborne pathogens to be detected, quantitated and/or monitored without requiring cell culture.   
     
     
         44 . A method for capturing live pathogens from a contaminating sample matrix by aptamer-based pathogen capture, comprising:
 reducing sodium concentration to less than 100 mM in pathogen capturing conditions;   reducing temperature to less than 20 degrees Celsius in pathogen capturing conditions; and   providing magnesium at a concentration greater than 0.1 mM and sufficient to increase aptamer melting temperatures to result in a stable secondary structure formation.   
     
     
         45 . The method of  claim 44 , further comprising applying a sample temperature by one or more cooling or heating elements to transfer the sample temperature to the sample prior to pathogen capture. 
     
     
         46 . A method for capturing live pathogens from a contaminating sample matrix by aptamer-based pathogen capture, comprising:
 reducing sodium concentration to less than 70 mM in pathogen capturing conditions;   reducing temperature to less than 20 degrees Celsius in pathogen capturing conditions; and   providing magnesium at a concentration greater than 0.25 mM and sufficient to increase aptamer melting temperatures to result in desired secondary structure formation.   
     
     
         47 . The method of  claim 46 , further comprising applying a sample temperature by one or more cooling or heating elements to transfer the sample temperature to the sample prior to pathogen capture. 
     
     
         48 . A method for detecting, quantitating and/or monitoring infectious particles, comprising:
 capturing one or more infectious particles from a contaminating matrix within a sample by aptamer-based capture or antibody/antigen capture; and   releasing the one or more captured infectious particles from aptamers or antibodies, thereby allowing the one or more infectious particles to be detected, quantitated and/or monitored without requiring cell culture.   
     
     
         49 . The method of  claim 48 , wherein the one or more infectious particles is one of bacteria, viruses, fungi, protozoa, worms, proteins, peptides and allergens. 
     
     
         50 . The method of  claim 48  or  claim 49 , wherein capturing one or more infectious particles comprises applying the sample to an isolation column containing multiple beads of one or more sizes. 
     
     
         51 . The method of  claim 50 , wherein bead surface of each of the multiple beads comprises a material that has been modified for aptamer or antibody attachment. 
     
     
         52 . The method of  claim 50  or  51 , wherein capturing one or more infectious particles further comprises applying the sample to a pre-filter container containing beads of same size or smaller than isolation column prior to the isolation column containing multiple beads of one or more sizes. 
     
     
         53 . The method of  claim 52 , wherein the beads of the pre-filter container have non-fouling surface properties. 
     
     
         54 . The method of any one of  claims 48 - 53 , further comprising washing the one or more captured infectious particles prior to releasing the one or more captured infectious particles. 
     
     
         55 . The method of any one of  claims 48 - 54 , wherein the one or more released captured infectious particles from aptamers or antibodies are concentrated prior to detection. 
     
     
         56 . The method of any one of  claims 48 - 55 , wherein the aptamer contains DNA, RNA, PNA, peptides or other natural or synthetic molecules. 
     
     
         57 . The method of any one of  claims 48 - 56 , further comprising nucleic acid extraction prior to detection. 
     
     
         58 . The method of  claim 57 , wherein detection contains nucleic acid detection comprising detecting DNA or RNA. 
     
     
         59 . The method of  claim 58 , wherein nucleic acid detection comprises performing polymerase chain reaction, isothermal amplification, hybridization detection, and/or sequencing. 
     
     
         60 . A system to automatically link a machine-readable code on a sample collection bag from a food industry sample to sample analysis results, comprising:
 a machine-readable code on the sample collection bag;   a machine-readable code on a bag used for paddle blending a sample;   a machine-readable code on a cartridge used for sample analysis;   a method to determine sample analysis results; and   a database to automatically link the machine-readable code on the sample collection bag to the sample analysis.   
     
     
         61 . The system of  claim 60 , wherein the sample collection bag and the bag used for paddle blending the sample are the same bag. 
     
     
         62 . The system of  claim 60  or  61 , wherein the machine-readable code on the cartridge optionally links to a type of analysis for that cartridge. 
     
     
         63 . The system of claim any one of  claims 60 - 62 , wherein the database to link the machine-readable code on the sample collection bag resides on a server accessible from a computer network. 
     
     
         64 . The system of  claim 63 , wherein metadata is contained in the database. 
     
     
         65 . The system of  claim 64 , wherein the metadata contains one or more information of sample information, sample collection information and cartridge information. 
     
     
         66 . The system of  claim 65 , wherein the metadata and machine-readable codes are used to determine whether the cartridge should be used to perform the analysis, including:
 determining type of pathogens or infectious agents the cartridge has been configured to analyze; and   determining viability of the cartridge, such as the age and whether the cartridge has been previously used.   
     
     
         67 . The system of any one of  claims 60 - 66 , wherein the food industry sample is one or more of a food product or a food processing environmental sample. 
     
     
         68 . The system of any one of  claims 60 - 66 , wherein the food industry sample is paddle blending with more than 25 milliliters of an aqueous solution prior to analysis. 
     
     
         69 . The system of  claim 68 , wherein the pathogen is one of bacteria, viruses, fungi, protozoa, worms, proteins, peptides and allergens from one or more of food, water, environment, soil, plant, animal, insect, or human. 
     
     
         70 . The system of  claim 68  or  69 , wherein digital data can be associated with one of cartridge manufacturing information, cartridge storage information, cartridge viability, cartridge lifetime, one or more pathogens the cartridge is designed for, the type of sample to be applied to the cartridge, data related to cartridge prior uses, and cartridge use data. 
     
     
         71 . The system of  claim 67 , wherein the cartridge digital data can be automatically written one or more of prior to analysis, during analysis or following analysis. 
     
     
         72 . A system for generating a graphical representation of analysis data, comprising a coordinate axis wherein a first and second axis of each data point can be correlated to sample collection location and number of pathogens at each location is represented by one or more of a third coordinate axis, values and one or more differences in color, hue or intensity. 
     
     
         73 . The system of  claim 72 , wherein sample collection location is one or more locations where samples are collected, such as where food is processed, prepared, stored, distributed, sold and/or consumed or a location where people are present, such as in locations where people congregate or work, or one or more locations of a farm, field or where animals are present.